Matrix-assisted laser desorption ionization (MALDI) is a technique suitable for an analysis of samples that barely absorb laser light or samples that will be easily damaged by laser light, such as protein. In this technique, a substance that is highly absorptive of laser light and easy to ionize is mixed beforehand into the sample, and this mixture is irradiated with laser light to ionize the sample. Particularly, mass spectrometers using the MALDI technique (which is hereinafter called the MALDI-MS) can analyze high molecular compounds having large relative molar masses without severely dissociating them. Moreover, mass spectrometers of this type are suitable for microanalysis. Due to these characteristics, the MALDI-MS has been widely used in recent years in biosciences and other fields.
In a MALDI-MS, reducing the spot size of the irradiation laser beam and relatively moving the spot on a sample provides an image that represents, for example, an intensity distribution of an ion having a specific mass (or two-dimensional distribution of a substance) on the sample. Such “imaging mass spectrometer” is expected to be particularly applicable, for example, in biochemical, medical and other fields to obtain distribution information of protein contained in biological cells (for example, refer to Non-Patent Document 1 and other documents).
In order to obtain useful information on a sample in the aforementioned application fields, it is desirable to perform the mass analysis with a high spatial resolution. The simplest yet most reliable method for improving the spatial resolution is to reduce the irradiation area of the laser beam so that the substance ionization can occur only within a small area. Normal types of MALDI-MS use a laser beam having a focused diameter of approximately several hundreds of μm, whereas the imaging mass spectrometer described in the aforementioned document uses a laser beam focused to be as small as approximately 30 μm in diameter. Furthermore, Non-Patent Document 2 and other documents disclose an example in which the laser beam was focused to a diameter of approximately 0.5 μm to obtain an image showing the substance distribution within a cell roughly several tens of μm in size. Due to such a high spatial resolution, these MALDI-MS systems can be used for a local analysis of a microsized area as well as for the determination of a one-dimensional or two-dimensional substance distribution.
In the case of performing a local analysis of a sample or obtaining a substance distribution image by means of, for example, an imaging mass spectrometer disclosed in the aforementioned documents, the sample is normally cut into a slice having a thickness from a few μm to several tens of μm and placed on a sample plate. Conventionally, the analysis process typically includes the following steps performed by an operator: removing the sample plate from the apparatus, placing a sample on the same plate, applying a matrix to the sample, and replacing the plate into the apparatus. Then, while observing the sample through a CCD camera or eyepiece, the operator specifies an analysis point or area by using the currently observed image (normally, a real-time image). Subsequently, a laser beam is delivered onto the specified point or area to perform the mass analysis.
The matrix, which is typically a solid, is subsequently dissolved in an organic solvent or the like, and the resultant matrix solution is placed on the sample. When the matrix solution is placed on the sample, a substance to be analyzed elutes from the sample into the solution. Subsequently, the solvent is vaporized to form a matrix crystal, with the aforementioned substance retained inside the crystal. Irradiating this crystal with a laser beam causes the ionization of the substance to be analyzed.
Various techniques have been proposed as a method for placing a matrix solution on a sample. One of the simplest methods is to drop a matrix solution of approximately several hundreds of nL onto a desired location. This operation can be performed with a commonly used manual pipetter and is therefore the simplest and inexpensive method. However, it has the drawback that the drop has such a large diameter (which is 2 to 3 mm for a drop of 500 nL) that the positional information of the substance to be analyzed will be lost after it elutes from the sample. This method is useful if a rough determination of the position suffices, but unsuitable for acquiring distribution information of a substance or performing a local analysis.
The most widely used method is to spray the matrix solution onto the sample. This method can uniformly place the matrix over a wide area of the sample and is suitable for acquiring substance distribution images. Due to the use of smaller droplets, the positional information is more precisely retained than in the aforementioned dropping method, so that the substance distribution image can be obtained with a high level of resolution.
Another conventional method includes discretely placing microsized droplets on a sample with an automatic pipetter. This method at least prevents the substance to be analyzed from moving between the neighboring droplets, so that the substance distribution image can be accurately produced. However, it is difficult to produce as small a droplet as in the spraying method, so that the spatial resolution of the substance distribution image cannot be equal to or higher than that achieved by the spraying method.
Regardless of which method is used, the matrix will eventually crystallize after the solution on the sample is dried. Although the matrix crystal is normally transparent, its observed image tends to be unclear due to the complex or fine shape of the crystal. FIGS. 13(a) and 13(b) are photographic images of a sample observed before and after a matrix solution is sprayed. The sample used in this example was a slice of a mouse's brain, onto which a CHCA solution was sprayed. As demonstrated in FIG. 13(b), the image of the sample surface becomes rather obscure after the matrix was applied. With such an unclear image, it is difficult to correctly select a specific area or point for the acquisition of a substance distribution image of a desired area on the sample or for a local analysis of a specific point on the sample.
Thus, the previously described imaging mass spectrometers using conventional MALDI techniques cannot always correctly perform the mass analysis of a desired point or area on a sample. Therefore, the user may possibly overlook really-required information or be forced to repeat the same analysis many times.    Non-Patent Document 1: Kiyoshi Ogawa et al., “Kenbi Shitsuryou Bunseki Souchi No Kaihatsu (Research and Development of Mass Microscope)”, Shimadzu Hyouron (Shimadzu Review), Shimadzu Hyouron Henshuu-bu, Mar. 31, 2006, Vol. 62, No. 3/4, pp. 125-135    Non-Patent Document 2: B. Spengler et al, “Scanning Microprobe Matrix-Assisted Laser Desorption Ionization (SMALDI) Mass Spectrometry: Instrumentation for Sub-Micrometer Resolved LDI and MALDI Surface Analysis”, Journal of American Society for Mass Spectrometry, 2002, Vol. 13, No. 6, pp. 735-748